Project Summary. The vast majority of breast cancer-related deaths are not caused by the primary tumor, but rather metastasis. Current studies mainly focus on the prevention of the early stages of metastasis, but since tumor cells have likely left the primary tumor at the time of diagnosis, inhibiting this aspect of metastasis may not be the most effective way to inhibit metastatic burden. Therefore, an emphasis on developing therapeutics against late-stage metastasis becomes essential. Six proteins are a family of transcription factors that are not only critical for development, but also for tumor progression and metastasis. Six2 is unique in that it specifically affects the later stages of breast cancer metastasis, demonstrating its potential as a therapeutic target to decrease metastatic burden. However, the molecular mechanisms that drive Six2-mediated metastasis remain largely unknown. Understanding the role of Six2 in this aspect of metastasis may provide insight into the development of new therapies targeting metastatic burden, particularly targeting metastatic establishment, outgrowth, and survival. Hypothesis: Six2 mediates the later stages of metastasis, particularly the establishment of tumors at secondary sites. Identification of genes regulated downstream of Six2 that mediate metastasis may enable more effective means to inhibit metastatic progression. Objective: By using stage- specific in vitro, ex vivo and in vivo models in combination with a comprehensive bioinformatics-driven candidate gene approach and shRNA screen, we will examine the role of Six2 in the later stages of metastasis. Specific Aims: 1) Define the later stage(s) of metastasis impacted by Six2. 2) Identify reversible targets regulated by Six2, and determine which of these downstream effectors are critical, and could possibly be therapeutic targets, in the later stages of metastasis. We will utilize cell lines that were isolated from the same mouse mammary primary tumor, representing triple negative breast cancer, but differing in their ability to complete the later-stages of metastasis. The 66cl4 cells express high levels of Six2 and are highly metastatic. The 4T07 cells express low levels of Six2, and can reach the lungs, but can only colonize when Six2 is introduced. We have engineered 66cl4 cells with Six2 knockdown (66cl4-Six2KD), 4T07 cells with Six2 overexpression (4T07-Six2), and 4T07 cells with inducible Six2 (4T07-Six2i) to assess the stage of metastasis in which Six2 is involved, using in vitro, ex vivo, and in vivo metastasis assays. To identify reversible Six2 targets, we have generated candidate gene lists from microarray data and will combine established roles from the literature, shRNA screens and genome-wide functional interaction network analysis to identify the most critical downstream effectors, which will be tested for their relevance in our in vitro, ex vivo, and in vivo assays. Finally, we will perform immunohistochemistry on patient tumor samples to determine whether Six2 regulates these targets in the setting of human breast cancer. This research will open up new avenues to explore for treatments to inhibit the later stages of metastasis, thus more effectively improving patient survival.